Constant and intermittent high glucose enhances endothelial cell apoptosis through mitochondrial superoxide overproduction

Abstract

Background

It has been previously shown that hyperglycemia enhances free radical production, inducing oxidative damage, which in its turn activates the death pathways implicated in cell apoptosis and necrosis. But the possible involvement of this pathway in the hyperglycemia-induced apoptosis of endothelial cells has not yet been reported.

Methods

To verify a possible connection between mitochondrial ROS production and apoptosis induced by both stable and oscillating high glucose, SOD, MnTBAP and TTFA was added to HUVEC cell culture medium. We measured nitrotyrosine and 8OHdG as oxidative stress parameters and Bcl-2 expression and Caspase-3 expression and activity as apoptosis indicators.

Results

Our results show that hyperglycemia, both stable or oscillating, increases oxidative stress and endothelial cell apoptosis through ROS overproduction at the mitochondrial transport chain level.

Conclusion

The prevention of mitochondrial oxidative damage seems to be a future important therapeutic strategy in diabetes. Copyright © 2006 John Wiley & Sons, Ltd.

Introduction

There is overwhelming evidence for the involvement of reactive oxygen species (ROS) in the pathogenesis of diabetes-associated vascular complications 1.

It has been previously shown that hyperglycemia enhances free radical production, inducing oxidative damage, which in its turn activates the death pathways implicated in cell apoptosis and necrosis 2–4.

In a previous study, we demonstrated that intermittent high glucose is more dangerous than constant high glucose medium for human umbilical vein endothelial cells (HUVECs) in culture, because in the former condition there was a marked increase in cellular apoptosis 5. Furthermore, we recently showed that the deleterious effect of intermittent glucose, as well as the effect of constant high glucose is also mediated by free radical over-generation 6

It was recently reported by Nishikawa and coworkers that the causal link between constant elevated glucose and hyperglycemic damage is the increased production of superoxide by the mitochondrial electron transport chain 7. A possible involvement of this pathway in the hyperglycemia-induced apoptosis of endothelial cells has not yet been reported. Therefore, the purpose of this study was to verify a possible connection between mitochondrial ROS production and apoptosis induced by both stable and oscillating high glucose. To this aim, HUVEC cell culture medium was enriched with Cu/Zn superoxide dismutase (SOD), or Mn(III)tetrakis(4-benzoic acid)porphyrin Chloride (MnTBAP) 6, a cell-permeable superoxide dismutase mimetic or thenoyltrifluoroacetone (TTFA), an inhibitor of mitochondrial complex II 7.

Materials and methods

Unless otherwise specified, reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Isolation and culture of human endothelial cells

HUVECs were isolated and pooled from umbilical cords obtained from normal vaginal deliveries by the procedure described by Jaffe et al.8. Cells were cultured in gelatin-coated 60 mm Petri dishes (Sarstedt) and grown in medium 199 (GIBCO BRL, Gaithersburg, MD, USA) supplemented with 20% heat inactivated fetal bovine serum (GIBCO), 25 µg/mL endothelial cell growth supplement, 90 µg/mL heparin (GIBCO), 0.25 µg/mL fungizone (GIBCO), 50 U/mL penicillin, and 50 U/mL streptomycin (GIBCO). The Petri dishes were incubated at 37 °C, in 5% CO₂ 95% air gas mixture. Primary cultures were fluid changed 24 h after seeding and were subcultured on reaching confluence by the use of 0.01% tripsin-EDTA, inactivated by dilution. Cultured cells were identified as endothelial by their morphology and the presence of factor VIII-related antigen detected using indirect immunofluorescence. Only first and second passage HUVECs were used in the study to avoid age-dependent cellular modifications. HUVECs were seeded at equal density (1.3 × 10⁵) in gelatin-coated 60 mm Petri dishes and allowed to attach overnight. They were then exposed to the experimental conditions for 14 days. Therefore, as in the previous experiment 6, three groups of cells were formed, each one receiving the following fresh media every 24 h: (1) continuous normal glucose medium (5 mM); (2) continuous high glucose medium (20 mM) and (3) normal and high glucose media alternating every 24 h. MnTBAP (100 microM) 6. SOD (Calbiochem, Darmstadt, Germany) 6 (100 units/mL), and TTFA (10 microM) 7 were added individually to the culture medium of each of the above-mentioned conditions.

Osmotic control was assured by incubating cells with 20 mM mannitol, both continuously or in alternating fashion.

Nitrotyrosine measurement

Nitrotyrosine content was evaluated by ELISA as previously reported 6. Briefly, an identical amount of protein from cell lysates (50 µg) was applied to a Maxisorp ELISA plate (NUNC Brand Products) together with nitrated BSA standard and allowed to bind overnight at 4 °C. After blocking, wells were incubated at 37 °C for 1 h with a mouse monoclonal antibody anti-nitrotyrosine (Upstate Biotechnology, Lake Placid, NY) (5 µg/mL) and then for 45 min at 37 °C with a peroxidase conjugated goat anti-mouse IgG secondary antibody diluted 1 : 1000. After washing, peroxidase reaction product was generated using TMB peroxidase substrate.

8-OHdG

8-Hydroxy-2′-deoxyguanosine amount in HUVEC digested DNA was determined using Bioxytech 8-OHdG-EIA Kit, a competitive enzyme-linked immunosorbent assay (ELISA) purchased from OXIS Health Products (Portland, OR, USA) following the manufacturer's instructions.

HUVECs DNA was isolated using DNAzol Reagent (Life Technologies, Grand Island, NY, USA) and quantified using a spectrophotometer.

Bcl-2 expression

Experiments were performed by western blotting analysis. Protein lysates were resolved by 12% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Buckinhamshire, UK). After the blocking step, the membrane was incubated at room temperature for 2.5 h with mouse anti-human Bcl-2 antibody (5 µg/mL) (Santa Cruz Biotecnology, California). Detection was performed using a secondary horseradish peroxidase-linked anti-mouse antibody (1 : 7000) (Santa Cruz Biotecnology, California) and enhanced chemioluminescence system (Pierce) using Kodak Bio Max Light-1 films. The intensity of the western blot signals was quantified by densitometry.

Bcl-2 ELISA

Bcl-2 amount was determined in HUVEC cell lysate using human Bcl-2 ELISA commercial kit purchased from Bender MedSystems (Wien, Austria) following the manufacturer's instructions.

Caspase-3 activity

Caspase-3 activity was determined using Caspase-3 Colorimetric Protease Assay Kit (Chemicon International Inc, Temecula, CA) following the manufacturer's instructions. Briefly, the kit is based on spectophotometric detection of the chromophore p-nitroaniline (pNA) after cleavage from the labeled substrate DEVD-pNA (DEVD is the sequence recognized by caspases). The free pNA can be quantified using a spectrophotometer or a microtiter plate reader at 405 nm. Comparison of the absorbance of pNA from an apoptotic sample with uninduced control allows determination of the fold increase in Caspase-3 activity.

Caspase-3 expression

Western blot was performed on cell lysate as previously described for Bcl-2. Proteins were resolved by 15% SDS-polyacrylamide gels electrophoresis and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Buckinhamshire, UK). After the blocking step, the membrane was incubated at room temperature for 1 h with rabbit anti-human polyclonal Caspase-3 antibody (1 : 200) (Santa Cruz Biotecnology, California). Secondary antibody was horseradish peroxidase-linked anti-rabbit (1 : 1000) (Santa Cruz Biotecnology, California). Detection was performed with enhanced chemioluminescence system (Pierce) using Kodak Bio Max Light-1 films. The intensity of the western blot signals was quantified by densitometry.

Statistical analysis

All data are means ± SD. Groups were compared using analysis of variance, and Bonferroni-Dunn's post hoc test was performed followed by Mann–Whitney U-test. The differences were considered significant at p < 0.05.

Results

The results of six different experiments for each experimental condition were analyzed through a 14-day period. Data from osmotic controls (20 mM mannitol) in any experimental condition were indistinguishable from normal glucose controls (data not shown).

Caspase activity and expression

After 14 days of experiment, Caspase-3 activity showed a great increase in stable high glucose condition, in comparison with normal glucose condition and this increase was even more marked in intermittent glucose condition. MnTBAP, SOD, and TTFA were all able to normalize the Caspase-3 increase previously observed, both in stable high glucose and in intermittent high glucose.

Caspase-3 expression, performed with western blot assay after 14 days of experiment, rose up in both stable and intermittent high glucose, but in the latter condition the increase was more marked. All added compounds produced a reduction in Caspase-3 expression both in cells cultured in stable high glucose and in those grown in intermittent glucose medium. These results are summarized in Figure 1.

Figure 1. (A) Graph showing Caspase-3 activity, N = 5 nM glucose; H = 20 nM glucose; H/L = 5/20 nM glucose. (B) Average densitometric quantification of Caspase-3 western blot. (C) Representative western blot for Caspase-3 protein. § = p < 0.01 normal versus high glucose; # = p < 0.001 intermittent versus normal glucose; * = p < 0.01 intermittent versus high glucose. Bars indicate ± SD

Bcl-2 expression

After 14 days of experiment, Bcl-2 ELISA, performed with a commercial kit and western blot analysis, showed that Bcl-2 protein concentration significantly decreased in the fluctuating condition, compared with either normal or stable high glucose condition. The adding of the inhibitor restores expression in both stable high and intermittent glucose. The results are summarized in Figure 2.

Figure 2. (A) Average densitometric quantification of Bcl-2 western blot. (B) Representative western blot for Bcl-2 protein. N = 5 nM glucose; H = 20 nM glucose; H/L = 5/20 nM glucose. (C) Bcl-2 ELISA of HUVEC lysates, N = 5 nM glucose; H = 20 nM glucose; H/L = 5/20 nm glucose. § = p < 0.01 normal versus high glucose; # = p < 0.001 intermittent versus normal glucose; * = p < 0.01 intermittent versus high glucose. Bars indicate ± SD

Nitrotyrosine and 8-OHdG measurement

After 14 days, when no oxidative stress inhibitory substance was added, the nitrotyrosine content increased in the 20 mM condition and more in the intermittent glucose condition in comparison with the 5 mM condition. The adding of the inhibitory substances equally inhibited the increase of nitrotyrosine in the 20 mM condition and in the intermittent glucose condition as regards the same conditions where no inhibitor was added (Figure 3). Similarly, after 14 days culture, the amount of 8-OHdG increased both in constant 20 mM glucose condition and in the fluctuating condition, but in the last one the content was more than doubled (Figure 3). The presence of the inhibitors blocked the 8-OHdG production in both conditions (Figure 3).

Figure 3. (A) Nitrotyrosine ELISA of HUVEC lysates, N = 5 nM glucose; H = 20 nM glucose; H/L = 5/20 nM glucose. (B) 8OHdG content in HUVEC DNA measured with ELISA technique. § = p < 0.01 normal versus high glucose; # = p < 0.001 intermittent versus normal glucose; * = p < 0.01 intermittent versus high glucose. Bars indicate ± SD

Discussion

Hyperglycemia causes many of the pathological consequences of both type 1 and type 2 diabetes 9, 10. Much of this damage is suggested to be a consequence of elevated production of ROS 1 and oxidative stress has recently been proposed as the unifying factor for the damaging effect of hyperglycemia 11.

Although in normal subjects plasma glucose concentration is strictly controlled within a narrow range, in diabetic patients plasma glucose concentration often changes markedly within a single day. It is now recognized that both hyperglycemia at 2 h during an oral glucose challenge, as well as glucose fluctuations per se are strong predictors of both cardiovascular disease 12 and microangiophatic complications 13. It has been suggested that these ‘hyperglycemic spikes’ may play a direct and significant role in the pathogenesis of vascular diabetic complications 14. Moreover, there is growing evidence that an acute increase of glycemia is accompanied by an oxidative stress generation 15 that may contribute to the generation of an endothelial dysfunction 15. Thus, clinical evidence suggests that in vivo glucose fluctuations may be dangerous for endothelial cells, and that this effect should be mediated by an oxidative stress.

In this study, we propose an experimental model in which primary cultures of human endothelial cells are exposed to intermittent high glucose, a condition that partly mimics what really happens in vivo in diabetic patients. In agreement with previous studies 2–4, we found that stable high glucose produced an increase in apoptosis and in oxidative stress generation. Moreover, according to our previous report 6, we confirm that intermittent glucose appears to worsen the pro-apoptotic effects of high glucose, thereby enhancing oxidative stress generation. Apoptosis is accompanied by an increase of both nitrotyrosine and 8-OHdG and is reversed by both SOD and the SOD mimetic MnTBAP, sustaining our previous finding. However, for the first time, we showed that inhibiting the mitochondrial electron transport complex II preserves endothelial cells from apoptosis induced by hyperglycemia, both stable and fluctuating. The effect of TTFA, a specific antioxidant active at mitochondrial level, in normalizing apoptosis, as well as nitrotyrosine and 8-OHdG, was equivalent to those of both SOD and MnTBAP suggesting therefore that an overproduction of free radicals at mitochondrial level is the mediator of the pro-apoptotic effect of hyperglycemia, stable or oscillating. Actually, MnTBAP and SOD were active against ROS originating in different districts inside the cell. The fact that a specific mitochondrial oxidative stress inhibitor achieves the same results suggests that the major source of ROS inside the cell, due to high glucose exposition, is the mitochondrial electron transport chain. To be specific, TTFA selectively inhibits mitochondrial complex II activity; our findings thus support Brownlee theory: inhibition of entrance of electron to Ubiquinone from Complex II blocks off ROS generation, preventing all downstream processes like apoptosis.

The overproduction of superoxide at mitochondrial level is emerging as a unifying explanation of the hyperglycemia-related diabetic complications 11, and studies confirm that this pathway works damaging the retina 16, the mesangial 17 and beta-cells 18 when exposed to high glucose. In this study, we showed that this pathway may also be involved in apoptosis of endothelial cells exposed to stable high glucose, and for the first time that it is also active in a condition of oscillating glucose. This last evidence may be of particular relevance, because cumulating in vitro evidence suggests that glucose variations may be more dangerous for the cells than stable high glucose. Mesangial cells cultured in periodic high glucose concentration increase matrix production more than the cells cultured in high stable glucose 19. Similarly, fluctuations of glucose display a more dangerous effect than stable high glucose on both tubulointerstitial cells and human renal cortical fibroblasts, in terms of collagen synthesis and cell growth 20, 21.

Our data suggest that the pathway involved in the damaging effect of intermittent high glucose on HUVECs is, at least in part, the same one working in stable high glucose concentrations. However, it appears noteworthy that it is enhanced in such conditions. At present, the molecular mechanisms specifically triggered on cultured HUVECs by periodically changing glucose concentrations are not known. A possible explanation may be that during chronic exposure to high glucose, some metabolic variations induced by this constant situation might change or feed back regulatory cell controls, partially counteracting the glucose toxic effect. Intermittent exposure to high glucose might reduce such adaptation, causing more pronounced toxicity.

Further studies are needed to better elucidate this point, even if it is growing the opinion that in diabetes the major source of damage is the rapid variation of blood glucose linked to the post-prandial state 22, 23. So, what is already known is that hyperglycaemia stimulates antioxidant enzymes synthesis 24 and that the decreased capacity of diabetic patients to respond to those repeated insults is tightly related to diabetic complications onset 25, 26. Our past and recent findings pointed out that exposition of endothelial 5 and retinal cells 27 to high oscillating glucose levels jeopardize the antioxidant response of the cell and increase levels of oxidative stress markers, leading to higher cell damage and apoptosis than exposition to high constant glucose. The next step will be to characterize what is missed, because to determine which regulatory pathway is compromised will be of great relevance in the prevention of diabetic complications.

Conclusions

In conclusion, our study has demonstrated that hyperglycemia, both stable or oscillating, increases endothelial cell apoptosis through ROS overproduction at the mitochondrial electron transport chain. Endothelial cell damage plays an important role in the pathogenesis of diabetic complication. Therefore, the prevention of mitochondrial oxidative damage seems to be a future important therapeutic strategy in diabetes 28.

Acknowledgements

This work was supported by an unrestricted grant from Novartis Pharma, Basel, Switzerland.